ABSTRACT: The four regulatory genes fscR1 to fscR4 in Streptomyces sp. strain FR-008 form a genetic arrangement that is widely distributed in macrolide-producing bacteria. Our previous work has demonstrated that fscR1 and fscR4 are critical for production of the polyene antibiotic candicidin. In this study, we further characterized the roles of the other two regulatory genes, fscR2 and fscR3, focusing on the relationship between these four regulatory genes. Disruption of a single or multiple regulatory genes did not affect bacterial growth, but transcription of genes in the candicidin biosynthetic gene cluster decreased, and candicidin production was abolished, indicating a critical role for each of the four regulatory genes, including fscR2 and fscR3, in candicidin biosynthesis. We found that fscR1 to fscR4, although differentially expressed throughout the growth phase, displayed similar temporal expression patterns, with an abrupt increase in the early exponential phase, coincident with initial detection of antibiotic production in the same phase. Our data suggest that the four regulatory genes fscR1 to fscR4 have various degrees of control over structural genes in the biosynthetic cluster under the conditions examined. Extensive transcriptional analysis indicated that complex regulation exists between these four regulatory genes, forming a regulatory network, with fscR1 and fscR4 functioning at a lower level. Comprehensive cross-complementation analysis indicates that functional complementation is restricted among the four regulators and unidirectional, with fscR1 complementing the loss of fscR3 or -4 and fscR4 complementing loss of fscR2 Our study provides more insights into the roles of, and the regulatory network formed by, these four regulatory genes controlling production of an important pharmaceutical compound.IMPORTANCE The regulation of antibiotic biosynthesis by Streptomyces species is complex, especially for biosynthetic gene clusters with multiple regulatory genes. The biosynthetic gene cluster for the polyene antibiotic candicidin contains four consecutive regulatory genes, which encode regulatory proteins from different families and which form a subcluster within the larger biosynthetic gene cluster in Streptomyces sp. FR-008. Syntenic arrangements of these regulatory genes are widely distributed in polyene gene clusters, such as the amphotericin and nystatin gene clusters, suggesting a conserved regulatory mechanism controlling production of these clinically important medicines. However, the relationships between these multiple regulatory genes are unknown. In this study, we determined that each of these four regulatory genes is critical for candicidin production. Additionally, using transcriptional analyses, bioassays, high-performance liquid chromatography (HPLC) analysis, and genetic cross-complementation, we showed that FscR1 to FscR4 comprise a hierarchical regulatory network that controls candicidin production and is likely representative of how expression of other polyene biosynthetic gene clusters is controlled.
